Model For Human Cytomegalovirus Infection Research Articles

Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence and Western blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.

Induction of an innate immune response and pathogenesis following ex vivo infection of human decidual tissues with human cytomegalovirus

Induction of an innate immune response and pathogenesis following ex vivo infection of human decidual tissues with human cytomegalovirus

Murine cytomegalovirus regulation of NKG2D ligands

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes morbidity risk in immunologically suppressed and immunodeficient patients including congenital infections. Approaches to curb the consequences of HCMV infections are restricted by a lack of complete understanding of viral pathogenesis. The infection of mice with murine cytomegalovirus (MCMV) as a model of HCMV infection has been particularly useful in elucidating the role of innate and adaptive immune response mechanisms. A large number of cytomegalovirus genes modulate the innate and the adaptive host immune response. The products of several MCMV genes are involved in subverting the natural killer (NK) cell response by down-modulating cellular ligands for the NKG2D receptor expressed on NK cells and CD8(+) T cells. Mutant viruses lacking these immunoevasion genes are attenuated with respect to virus growth in vivo. Given the importance of the NKG2D receptor in controlling both NK- and T cell-mediated immunity, it is of tremendous importance to understand the molecular mechanisms and consequences of viral regulation of the NKG2D ligands.

A model of human cytomegalovirus infection in severe combined immunodeficient mice

Animal models for the evaluation of therapies against human cytomegalovirus (HCMV) are limited due to the species-specific replication of CMV. Models utilizing human fetal tissues implanted into SCID mice have been used but tend to be labor intensive and require human tissues. We therefore developed a model using HCMV-infected human foreskin fibroblasts (HFF) seeded onto a biodegradable gelatin matrix (Gelfoam). Infected HFFs are then implanted subcutaneously into SCID mice. We next evaluated two antivirals in this model. Treatment from days 0 to 5 with ganciclovir (GCV) produced a marginally significant reduction in viral titer while treatment from days 0 to 14 resulted in a more significant reduction in viral titers of 1.47 log 10 pfu/ml ( P < 0.0001). Viral titers were similarly reduced in a group receiving GCV treatment from days 7 to 14 post-implantation (1.50 log 10 pfu/ml, P < 0.0001). Cidofovir therapy from days 7 to 14 reduced viral titers by almost 2 log 10 pfu/ml (from 3.51 ± 0.31 log 10 pfu/ml in untreated animals to 1.56 ± 0.40 log 10 pfu/ml in treated animals, P < 0.0001). These results indicate that the Gelfoam-HCMV SCID mouse model is suitable for the in vivo evaluation of new antivirals against HCMV and is simpler and more convenient than previous models.

Infection with rat cytomegalovirus (CMV) in the immunocompromised host is associated with the appearance of a T cell population with reduced CD8 and T cell receptor (TCR) expression.

Infection with human cytomegalovirus (HCMV) mostly results in a chronic subclinical infection; the immune system is unable to eliminate the virus and is apparently in equilibrium with the persistent virus. In the immunosuppressed host this equilibrium is disturbed, resulting in clinical infection. Rat cytomegalovirus (RCMV) infection in its host can be used as a model for HCMV infection. Using flow cytometry we examined the effect of acute RCMV infection on the composition of leucocyte subsets in the peripheral blood of both immunocompetent and immunosuppressed (5 Gy total body irradiation) Lewis rats. Special attention was paid to the natural killer (NK) cells and the CD8+ T cells known to be involved in the control of viral infections. Furthermore, we determined the presence of leucocyte subsets in the internal organs by immunohistochemistry. In immunocompetent rats, infection caused a small increase in NK cells and a large increase in CD8+ T cells. In contrast, infection of immunosuppressed rats caused a marked increase in NK cells and a small increase in CD8+ T cells, consisting of T cells with reduced expression of both CD8 and TCR. This phenomenon is characteristic of anergic CD8+ T cells, possibly explaining the ability of the virus to escape elimination by the immune system. The increase of NK cells in the peripheral blood of immunosuppressed, RCMV-infected rats could also be detected in kidney, liver, lung and pancreas, but not in salivary gland. This could explain the long persistence of infectious virus in the salivary gland.

Unrestricted replication of human cytomegalovirus in hydrocortisone-treated macrophages.

Monocytes differentiated in the presence of phytohemagglutinin P-stimulated T cells could be infected with human cytomegalovirus AD169 and produced low levels of infectious virus. Additional treatment with therapeutic levels of hydrocortisone resulted in a 10- to 100-fold increase in infectious virus production. Hydrocortisone-treated cells demonstrated immediate-early protein kinetics similar to that observed with human fibroblasts, whereas a delay of up to 24 h was observed with untreated cells. Late protein production was barely detectable by immunostaining without hydrocortisone treatment. In treated cells, however, late protein was detected and the levels correlated with the number of cells producing infectious virus. This system provides a model for human cytomegalovirus infection of macrophages in humans.

Ganciclovir

Ganciclovir is a nucleoside analogue with antiviral activity in vitro against members of the herpes group and some other DNA viruses. It has demonstrated efficacy against human cytomegalovirus infections and should be considered a first-line therapy in the treatment of life- or sight-threatening cytomegalovirus infection in immunocompromised patients. Clinical efficacy varies with the underlying aetiology of immunocompromise and the site of disease, and prompt diagnosis and early treatment initiation appear to improve the response. In patients with cytomegalovirus pneumonia, particularly bone marrow transplant recipients, concomitant administration of cytomegalovirus immune globulin may significantly improve clinical outcome. Maintenance therapy to prevent recurrence is usually required by bone marrow transplant recipients until the recovery of adequate immune function, whereas AIDS patients may require indefinite ganciclovir maintenance therapy to prevent disease progression, as ganciclovir (like other antivirals) does not eradicate latent viral infection. Haematological effects occur relatively frequently during ganciclovir administration but are usually reversible. Ganciclovir has not been directly compared with other antiviral drugs because of the absence until recently of other effective treatments. However, comparative studies with foscarnet, particularly in cytomegalovirus retinitis, will be of considerable interest. Thus, ganciclovir represents a major advance in the therapy of severe cytomegalovirus infections in immunocompromised patients. Comparative studies, and investigation of ways of reducing toxicity (intravitreal administration; concomitant use of stimulants of haematopoiesis; use in conjunction with other antivirals with differing mechanisms of action), may further expand its eventual role.

Immunoblot Analysis of the Antibody Response to Murine Cytomegalovirus in Genetically Resistant and Susceptible Mice

The murine of human cytomegalovirus infection was employed to analyse the kinetics of antibody production to murine cytomegalovirus (MCMV) structural and immediate early (IE) polypeptides following MCMV infection of genetically resistant and susceptible strains of mice. A total of 22 structural and six non-structural. IE proteins were identified. Analysis of immunoblots by densitometry identified four patterns of antibody reactivity to MCMV structural polypeptides during primary and secondary antibody responses over a period of 5 weeks post-infection (p.i.). Firstly, antibodies were strongly reactive with an 83K protein soon after infection, with levels which decreased with time: antibodies to a second group of viral proteins were also recognized soon after infection, but consistent levels of reactivity were maintained. Viral proteins that were recognized beyond day 14 p.i. or following a second MCMV infection formed the third group; the fourth consisted of viral proteins that were detected at variable times p.i. by antisera different mouse strains. The kinetics and intensity of the antibody response to individual viral proteins were influenced by virus dose, time p.i. and by a second MCMV infection. In addition, the genetic constitution of the host influenced the antibody response to MCMV proteins both quantitatively and qualitatively. In particular, sera from mice possessing C57BL but not BALB/c genes detected a 56K Mr viral during seroconversion. Antibody reactivity to this protein was shown to segregate among sera from CXB mice, with a strain distribution pattern which indicated a linkage with the b locus on chromosome 4. Finally, expression of the 56K protein was detected in B10. BR but not BALB/c embryo fibroblasts in vitro, with expression being a dominant trait in (BALB/c x B10.BR) F1 embryo fibroblasts. Thus, host genes may influence the expression of this structural viral protein.